Biomod/2011/MIT/Origami/HIV Seek and Destroy

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HIV Life Cycle

HIV's life cycle proceeds in 3 stages: entry to the cell, replication and transcription, and finally assembly and release. With the exception of the initial stage, our research into the other 3 stages of the life cycle is currently incomplete, and although we know the major events that occur at each stage, the particular order in which they occur might not be exactly as detailed, although essentially correct.

Entry to the Cell

One of the curious things about HIV as a virus is that it only has the ability to infect cells that express the surface protein CD4, which is also what makes it so dangerous. CD4 is expressed only on certain immune cells, including macrophages, dendritic cells, and helper T cells. Its natural function is to interact with MHCII on certain antigen presenting cells and act as a crucial mediator of the immune response. HIV however uses it's own glycoprotein120 (gp120) to interact with CD4 and induce a conformation change that leaves a second co-receptor expressed which HIV interacts with using a second glycoprotein, glycoprotein41(gp41), which is anchored to the cell membrane, while the gp120 is only noncovalently bonded to gp 41.

The co-receptor in question can be one of two, either chemokine receptor type 5(CCR5) or chemokine receptor type 4(CXCR4). Most strains of HIV use CCR5 exclusively as their co-receptor, some can use either, and very few use CXCR4 exclusively. Once the co-receptor and receptor interactions are in place, gp41 undergoes a change that allows it to fuse the it's own membrane with the membrane of the target cell. The viral capsid and associated proteins are then able to enter the cell and begin the process of replication.

Replication and Transcription

Once HIV is inside the cell, a protein called reverse transcriptase(RT) begins to take HIV's RNA genome and transcribe it into a complementary DNA(cDNA) equivalent version. RT functions in a manner similar to DNA polymerase, which is used to replicate DNA, but it uses an RNA as a template for the new strand. Once the synthesis of the viral cDNA is completed, another viral enzyme, integrase, takes this cDNA and inserts it into the cellular DNA. At this point, the cell becomes a permanent carrier of the viral genome.

Research has been done in an attempt to learn more about the details of the reverse transcription complex that HIV uses. Currently, it seems that a protein called Tat is required for reverse transcription, gene expression, and proliferation of the virus. It is thought to do this by binding to the tar sequence of RNA. Tat is also released by infected cells (it can cross the cell membrane) and seems to recruit HIV to uninfected cells. More on this later. Also, the immunogenic regions of Tat are conserved through different strands of HIV, making it a reliable target for a vaccine, or to be used in a vaccine. Research is currently being done to develop vaccines based on Tat [1]

Other research suggests that certain cell factors stabilize the reverse transcription complex, and also aid DNA strand transfer and DNA synthesis. Very recent research is trying to determine exactly what these factors are, and many seem to be various cellular proteins. Some research has determined a couple cellular components that do not seem to affect the complex. We may not have enough detail to use this concept, but we can move forward with the understanding that to allow for efficient transcription, the cell environment should not be disturbed.

Once the viral genome is integrated into the cellular genome, the expression of viral genes begins. Presumably a certain amount of transcription factors are needed to stimulate the particular expression of these genes, but our research has yet to reveal whether these factors are carried in the capsid with the virus itself. This will be updated when we find out. Nevertheless, the synthesis of certain important proteins, such as the capsid envelope and the glycoproteins(which are actually synthesized as one polypeptide, glycoprotein 160, and then later cleaved by a viral protease) starts when the transcription begins

Assembly and Release

One of the more important events of the last third of HIV's life cycle is the cleaving of gp160. A viral protease breaks the polypeptide into the functional subunits gp120 and gp41 that are used to enter the host cell. Also of particular importance is how the viral proteins and capsid are transported to the cell membrane. It is known that on their way in, they travel along the cells own actin filaments, so it is probable that they do the same on the way out, but the literature we've read so far has not revealed it beyond a shadow of a doubt. At the earliest stages of assembly, the interactions between CA protein domains of the Gag polyprotein help drive the formation of immature particles at the membrane of host cells. Once they are released, the Gag polyprotein undergoes proteolytic processing, leading to capsid assembly and the formation of the mature virus. After fusion, CA undergoes a controlled disassembly reaction so that the viral genome can be transcribed properly. The protease inhibition is a target of some drugs, and blocking the localization of the proteins to the cellular membrane is a possible target as well. The proper folding of the viral proteins into the capsids is also a possible target. It is probably more difficult since the literature suggests that the mechanisms of folding are not completely understood. On its way out, the virus takes a piece of the cell's membrane, studded with HIV glycoproteins, to infect other cells.

Available Treatments

There are currently three main classes of drugs that are used to treat HIV infection, all with different mechanisms of action. There are the reverse transcriptase inhibitors, the entry/fusion inhibitors and the protease inhibitors. There is also another class of drugs called integrase inhibitors, but there is only one type of this drug approved by the FDA and it is relatively new, and there is research being conducted on another class, the maturation inhibitors, but for the moment we'll focus on the first three classes.

Reverse Transcriptase Inhibitors

Among these, there are two classes, the Nucloside analog reverse-transcriptase inhibitors(NRTIs) and the Non-nucleoside reverse-transcriptase inhibitors(NNRTIs). NRTIs are similar in function and structure to natural nucleotides, and they compete with nucleotides to be added to the growing cDNA chain during reverse transcription, but since they lack a 3' OH, their incorporation into the strand causes a chain termination. NNRTIs on the other hand are allosteric inhibitors of reverse transcriptase, they bind to domains of the enzyme that hinder conformational changes required for adequate polymerization of the growing DNA strand. The problem with these is that they bind to a less conserved pocket of the p66 subunit(RT consists of a p66 and p51 subunits) which may make it more likely for the virus to develop resistance.

Entry/Fusion Inhibitors

There are two of these types of drugs that are FDA approved, enfuvirtide and maraviroc, each with it's own target. Maraviroc is an allosteric modulator of CCR5. It appears to cause changes in shape that inhibit the ability of viral glycoproteins to interact with it. It would therefore have no effect on strains of HIV that use CXCR4 as a coreceptor, but 80% of HIV strains use CCR5. Enfuvirtide acts by mimicking normal HIV-cell interactions to prevent said interactions from actually occurring. It binds to gp41 to prevent it from fusing the viral and cellular membranes. Downsides of Enfuvirtide include it's cost(around 25,000 per year in the US) and the fact that it is not available in an orally administered form, it must be injected. It is generally used only when a patient has developed resistance to other drugs, because the regimen is difficult to follow.

Protease Inhibitors

Protease is responsible for cutting the newly synthesized proteins at the appropriate places to create the mature proteins of the HIV virus. HIV protease inhibitors (PI) block the HIV aspartyl protease, which cleaves the HIV gag and gag-pol polyprotein backbone at nine specific cleavage sites. Most PIs are designed to mimic a phenylalanine - proline peptide bond because three of the nine cleavages occur between a phenylalanine or a tyrosine and a proline. It has been found that PIs also inhibit inflammatory cytokine production and modulate antigen presentation and T-cell responses.


Target Ideas

GP120

Capsid

Tat Protein

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